Regulatable growth of filamentous fungi

ABSTRACT

The present invention generally relates to hyphal growth in fungi and in particular describes the modulation of genes associated with hyphal growth in filamentous fungi. The present invention provides methods and systems for the production of proteins and/or chemicals from filamentous fungi which comprise modulation of genes associated with hyphal growth. Specifically, the present invention is directed to a full length cotA gene, its gene product and methods of use.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a Continuation of U.S. patent applicationSer. No. 10/100,252, filed Mar. 14, 2002, now issued U.S. Pat. No.6,936,449, which claims priority to 60/276,571 filed Mar. 15, 2001 andto 60/276,618, filed Mar. 14, 2001.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not Applicable.

BACKGROUND OF THE INVENTION

Growth morphology is an important factor affecting fermentation offilamentous fungi during production of proteins and fine chemicals.cot-1 of Neurospora crassa is a colonial temperature sensitive mutationthat has been described in detail Steele, et al., Arch. Microbiol.113:43 (1977) and Collinge, et al., Trans. Br. Mycol. Soc. 71:102(1978)). Germination and growth of the mutant is normal at 26° C., but ashift to 37° C. causes the cessation of hyphal tip extension, andemergence of lateral branches at an abnormally high frequency to givehyperbranching germlings. An increase in the frequency of septation isalso seen. Sequence analysis indicated the gene product belongs to thefamily of serine/threonine protein kinases (Yarden, et al., EMBO J.11:2159 (1992). These kinases act in signal transduction pathways, buthow cot-1 is integrated into the pathway(s) controlling hyphal growthpolarity has yet to be elucidated. The specific mutation that causes thetemperature sensitivity in N. crassa cot-1 has been found to be ahistidine to arginine substitution (Gorovits, et al., Fungal Geneticsand Biol. 27:264 (1999).

There remains a need in the art for genes that control growth morphologyin filamentous fungal cells, like Trichoderma and Aspergillus, that areused as a source of recombinant proteins in an industrial setting and toenhance the production of proteins and fine chemicals. This inventionmeets this need as well as others.

SUMMARY OF THE INVENTION

One embodiment of this invention provides for an isolated polynucleotideselected from the group consisting of a nucleic acid sequence thatencodes or is complementary to a sequence that encodes a cotApolypeptide having at least 85% sequence identity to the amino acidsequence presented in any one of FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ IDNO:4) or FIG. 6 (SEQ ID NO:6); a nucleic acid sequence that encodes oris complementary to a sequence that encodes a cotA polypeptide having atleast 90% sequence identity to the amino acid sequence presented in anyone of FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4) or FIG. 6 (SEQ IDNO:6); a nucleic acid sequence that encodes or is complementary to asequence that encodes a cotA polypeptide having at least 95% sequenceidentity to the amino acid sequence presented in any one of FIG. 2 (SEQID NO:2), FIG. 4 (SEQ ID NO:4) or FIG. 6 (SEQ ID NO:6); a nucleic acidsequence that encodes or is complementary to a sequence that encodes acotA polypeptide having the amino acid sequence presented in any one ofFIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4) or FIG. 6 (SEQ ID NO:6); thenucleic acid sequence presented as any one of SEQ ID NOs:1, 3 or 5 (FIG.1, 3 or 5, respectively) a portion greater than 200 bp thereof, or thecomplement thereof, and a nucleic acid sequence that hybridizes, underhigh stringency conditions to the sequence presented as any one of SEQID NOs:1, 3 or 5, or the complement or a fragment thereof, wherein saidisolated polynucleotide, when induced in a fungal cell, causes said cellto grow more slowly.

In a first aspect of this embodiment, the % identity is calculated usingthe CLUSTAL-W program in MacVector version 6.5, operated with defaultparameters, including an open gap penalty of 10.0, an extended gappenalty of 0.1, and a BLOSUM 30 similarity matrix.

In a second aspect of this embodiment, hybridization is conducted at 42°C. in 50% formamide, 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100μg/ml denatured carrier DNA followed by washing two times in 2×SSPE and0.5% SDS at room temperature and two additional times in 0.1×SSPE and0.5% SDS at 42° C. In yet another embodiment, the isolatedpolynucleotide is an RNA molecule.

In a third aspect of this embodiment, the isolated polynucleotide isoperably linked to a regulatable promoter. In a preferred aspect of thisembodiment the promoter is induced by maltose in the fungal cellenvironment. In another preferred aspect of this embodiment, thepolynucleotide is in the antisense orientation.

In a fourth aspect of this embodiment, the polynucleotide is SEQ IDNO:1.

In a fifth aspect of this embodiment, the polynucleotide is SEQ ID NO:3.

In a sixth aspect of this embodiment, the polynucleotide is SEQ ID NO:5.

In second embodiment of this invention, a recombinant filamentous fungalhost cell comprising a cotA polynucleotide is provided. In one aspect ofthis embodiment, the fungal host cell is a member of Aspergillus spp. Inanother aspect of this embodiment, the cell is an Aspergillus nigerfungal cell. In yet another aspect of this invention, the cell is amember of Trichoderma, more preferred is T. reesei. In further aspect ofthis embodiment, the recombinant fungal host cell is transformed withthe vector comprising any one of SEQ ID NOs:1, 3 or 5 operably linked toa regulatable promoter. In a particularly preferred aspect of thisembodiment, the vector integrates into the wild-type cotA gene. Inanother aspect of this embodiment, the vector integrates ectopically. Inan aspect of this embodiment, the polynucleotide integrates in theantisense orientation.

In a third embodiment of this invention, a substantially purified cotApolypeptide with the biological activity of a serine/threonine kinase isprovided. The biologically active polypeptide comprises a sequenceselected from the group consisting of an amino acid sequence having atleast 85% sequence identity to the amino acid sequence presented in anyone of FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4) or FIG. 6 (SEQ IDNO:6); an amino acid sequence having at least 90% sequence identity tothe amino acid sequence presented in any one of FIG. 2 (SEQ ID NO:2),FIG. 4 (SEQ ID NO:4) or FIG. 6 (SEQ ID NO:6); an amino acid sequencehaving at least 95% sequence identity to the amino acid sequencepresented in any one of FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4) orFIG. 6 (SEQ ID NO:6); the amino acid sequence presented as any one ofSEQ ID NOs:2, 4 or 6, a substantially purified biologically activefragment of the amino acid sequence presented as any one of SEQ ID NO:2,4 or 6, and a substantially purified full length protein comprising theamino acid sequence encoded by either of SEQ ID NOs:13 or 14.

In a fourth embodiment of this invention, a purified antibody thatspecifically binds to a cotA polypeptide is provided. In one aspect ofthis embodiment, a polynucleotide is provided that encodes a cotApolypeptide that specifically binds to an antibody.

In a fifth embodiment a method is provided for the detection of apolynucleotide that encodes a filamentous fungal cotA in a biologicalsample. The method includes, but is not limited to, the following steps:(a) hybridizing, under moderate stringency, to a nucleic acid materialof said biological sample, a polynucleotide fragment derived from anyone of the sequences identified as SEQ ID NOs:1, 3 or 5, the fragmenthaving a length of between about 15 and 250 nucleotides, thereby forminga hybridization complex; and (b) detecting said hybridization complex;wherein the presence of said hybridization complex correlates with thepresence of a polynucleotide encoding the cotA protein in saidbiological sample. In a first aspect of this embodiment, the fragment isbetween 15 and 30 nucleotides in length. In another aspect, the fragmentis between 30 and 100 nucleotides in length. In yet another aspect, thefragment is between 100 and 200 nucleotides in length, more preferred isa fragment between 200 and 250 nucleotides. In a final aspect, thefragment is about 241 nucleotides in length. In a second aspect of thisembodiment, the biological sample is a filamentous fungal cell lysate.In third aspect of this embodiment, an agonist of cotA protein isidentified. The method comprises the steps of (a) transfecting a fungalhost cell with a polynucleotide that encodes a cotA protein; (b)inducing the expression of cotA; (c) contacting a test compound with theso induced fungal host cell, (d) measuring the effect of the testcompound on the growth of the induced fungal cell; and (e) identifyingthe test compound as a candidate compound if it modulates the growth ofthe fungal cell beyond a selected threshold level.

In a final embodiment of this invention, a method of inducing a compactgrowth morphology of a filamentous fungal host cell is provided. In apreferred aspect of this embodiment, the fungal cell is a member of theTrichoderma genus, most preferred is Trichoderma reesei. In a morepreferred aspect of this embodiment, the fungal cell is a member of theAspergillus genus. In a most preferred aspect, the fungal cell is a A.niger cell. The method comprises the steps of transfecting said fungalhost cell with a cotA polynucleotide or a fragment thereof operablylinked to an inducible promoter, and exposing the transfected fungalhost cell to a compound that induces expression of the cotApolynucleotide. In another preferred aspect of this embodiment, the cotApolynucleotide is as shown in any one of SEQ ID NOs:1, 3, 5, 13 or 14.In a particularly preferred aspect of this embodiment, the cotApolynucleotide is in the antisense orientation. In another particularlypreferred aspect, the promoter is inducible by maltose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the nucleic acid sequence of the truncated Aspergillus nigercotA of this invention (SEQ ID NO:1).

FIG. 2 shows the deduced amino acid sequence of the truncatedAspergillus niger cotA of this invention (SEQ ID NO:2).

FIG. 3 is the nucleic acid sequence of the truncated Aspergillusnidulans cotA of this invention (SEQ ID NO:3).

FIG. 4 shows the deduced amino acid sequence of the truncatedAspergillus nidulans cotA of this invention (SEQ ID NO:4).

FIG. 5 is the nucleic acid sequence for an internal cotA fragment fromTrichoderma reesei (SEQ ID NO:5).

FIG. 6 is the deduced amino acid sequence for the internal cotA fragmentfrom Trichoderma reesei (SEQ ID NO:6).

FIGS. 7A-C are a sequence alignment of cotA and related kinases. spo1 isthe truncated cotA of Aspergillus niger. andcot is the full length cotAfrom Aspergillus nidulans. COT1-NEUCR is the full length cot-1 fromNeurospora crassa. S70706 is from Colletotrichum trifolli. KNQ1_YEAST isfrom S. cerevisiae. DMK_HUMAN is human myotonic dystrophy kinase.

FIG. 8A is a schematic of the integration of the expression vector,pSMB5, into the cotA locus of Aspergillus niger. FIG. 8B is a schematicof the locus after transformation.

FIG. 9. Comparison of wt and glaAp-cotA strains on a variety ofnon-repressing (maltose) and repressing (xylose) carbon sources. Grownuntil same morphological age then stained with calcoflour. Bands=10 μm.YEPX Yeast Extract, Peptone and Xylose. YEPD Yeast Extract, Peptone andGlucose.

FIGS. 10A and 10B are photographs of Aspergillus niger transfected withcotA in the antisense orientation under the control of the glaApromoter. As can be seen, a slowed growth phenotype is observed whentransformed cells are grown in the presence of xylose or maltose (FIGS.10 a and b).

FIG. 11 is the nucleic acid sequence of the full length cotA fromAspergillus niger (SEQ ID NO: 13). All introns are underlined. The startcodon is in bold type. The functional truncated cotA gene ends at theitalicized, underlined codon and is at the beginning of the secondintron. The stop codon for the full-length cotA is shown in bold type.

FIG. 12 is the nucleic acid sequence of the full length cotA fromAspergillus nidulans (SEQ ID NO: 14).

FIG. 13 is a 269 bp probe from Aspergillus niger (SEQ ID NO:15).

FIG. 14 is the deduced amino acid sequence of the full-lengthAspergillus niger cotA of this invention (SEQ ID NO:16).

DETAILED DESCRIPTION OF THE INVENTION

Many proteins and other compounds with industrial or pharmaceutical use,e.g., cellulases, proteases, lipases, xylanases, are produced infilamentous fungal cell cultures. An ongoing problem is that as thefungal cells divide and the culture expands, the number of cells in theculture make the culture viscous. In a continuous culture, oxygen andother nutrients do not mix as readily and are therefore unavailable forall the cells. In a batch culture, nutrients are exhausted at a fasterrate as the culture expands. In both cases, the growth of the culture aswell as the production of the desired protein reaches a plateau andbegins to drop. It has been found that transforming filamentous fungalcells with cotA-encoding nucleic acids under the control of aregulatable promoter causes the transformed cells to reduce the rate ofgrowth when in the presence of a compound that regulates the promoter.Transformation can occur with the cotA-encoding nucleic acid integratingin the cotA locus or ectopically. The reduced growth phenotype is seenin both instances. Without being held to any theory, it is believed thatif integration occurs in the cotA locus, expression of wild type cotA isunder the control of the heterologous and regulatable promoter andbecomes inducible.

Fungal protein synthesis is located at the fungal growing tips.Increasing the number of growing tips by isolating hyperbranchingmutants has benefits in fermentation. The compact morphology seen inhyperbranching mutants such as cot-1 would be useful in fungalfermentations where reduced viscosity could allow better fermentationperformance. Not to be limited by theory, it is believed that the lowviscosity of the fermenation mixture allows for better oxygenation ofthe media, which in turn enhances cell protein production.

A temperature sensitive cotA mutant may be created in various ways. Forexample, putting the cotA gene under a temperature sensitive promoter orcreating a temperature sensitive cotA mutant in the filamentous fungicotA homolog similar to the N. crassa cot-1 variant would be especiallydesirable. In an embodiment the filamentous fungi cotA homolog has beenaltered to have a substitution corresponding to the histidine toarginine substitution found in the N. crassa cot-1 variant. Thus, atemperature sensitive mutant that produces a hyperbranching phenotypewith a compact morphology at a higher temperature is particularlydesirable.

In one embodiment the endogenous cotA gene is replaced with atemperature sensitive cotA mutant having a substitution at the histidineresidue that corresponds H352 in N. crassa. In one aspect the alterationis a substitution of the histidine to arginine (as found in thetemperature sensitive N. crassa cot-1 variant). Thus, once thetemperature sensitive cotA mutant has integrated into the host genome byhomologous recombination it will be under the regulation of theendogenous cotA control sequences.

The ability of cotA mutant to effect protein secretion may be examinedby growing the cotA mutant on petri dishes with starch as the solecarbon source. Manipulation of the expression of the cotA gene productwould have utility in increasing heterologous protein secretion.

I. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All references are incorporatedby reference for all purposes. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described. For purposes of the present invention, thefollowing terms are defined below.

The term “polypeptide” as used herein refers to a compound made up of asingle chain of amino acid residues linked by peptide bonds. The term“protein” as used herein may be synonymous with the term “polypeptide”or may refer, in addition, to a complex of two or more polypeptides. AcotA polypeptide includes, but is not limited to, a polypeptide encodedby the cotA polynucleotides of this invention. Specifically, cotApolypeptides or proteins encompass Aspergillus and Trichoderma cotA fulllength proteins, including, but not limited to, signal or leadersequences, mature proteins and fragments thereof.

As used herein, the term “overexpressing” when referring to theproduction of a protein in a host cell means that the protein isproduced in greater amounts than its production in its naturallyoccurring environment.

As used herein, the phrase “protein associated with hyphal growth”refers to a protein which is capable of modulating hyphal growth infungus. Illustrative of such proteins are the cotA proteins disclosedherein.

The term “nucleic acid molecule” includes RNA, DNA and cDNA molecules.It will be understood that, as a result of the degeneracy of the geneticcode, a multitude of nucleotide sequences encoding a given proteins suchas cotA may be produced. The present invention contemplates everypossible variant nucleotide sequence, encoding cotA, all of which arepossible given the degeneracy of the genetic code. A “heterologous”nucleic acid construct or sequence has a portion of the sequence whichis not native to the cell in which it is expressed. Heterologous, withrespect to a control sequence refers to a control sequence (i.e.promoter or enhancer) that does not function in nature to regulate thesame gene the expression of which it is currently regulating. Generally,heterologous nucleic acid sequences are not endogenous to the cell orpart of the genome in which they are present, and have been added to thecell, by infection, transfection, microinjection, electroporation, orthe like. A “heterologous” nucleic acid construct may contain a controlsequence/DNA coding sequence combination that is the same as, ordifferent from a control sequence/DNA coding sequence combination foundin the native cell.

As used herein, the term “vector” refers to a nucleic acid constructdesigned for transfer between different host cells. An “expressionvector” refers to a vector that has the ability to incorporate andexpress heterologous DNA fragments in a foreign cell. Many prokaryoticand eukaryotic expression vectors are commercially available. Selectionof appropriate expression vectors is within the knowledge of thosehaving skill in the art.

Accordingly, an “expression cassette” or “expression vector” is anucleic acid construct generated recombinantly or synthetically, with aseries of specified nucleic acid elements that permit transcription of aparticular nucleic acid in a target cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid sequence to betranscribed and a promoter.

As used herein, the term “plasmid” refers to a circular double-stranded(ds) DNA construct used as a cloning vector, and which forms anextrachromosomal self-replicating genetic element in many bacteria andsome eukaryotes.

As used herein, the term “selectable marker-encoding nucleotidesequence” refers to a nucleotide sequence which is capable of expressionin fungal cells and where expression of the selectable marker confers tocells containing the expressed gene the ability to grow in the presenceof a corresponding selective agent.

As used herein, the term “promoter” refers to a nucleic acid sequencethat functions to direct transcription of a downstream gene. Thepromoter will generally be appropriate to the host cell in which thetarget gene is being expressed. The promoter together with othertranscriptional and translational regulatory nucleic acid sequences(also termed “control sequences”) are necessary to express a given geneor nucleic acid sequence. In general, the transcriptional andtranslational regulatory sequences include, but are not limited to,promoter sequences, ribosomal binding sites, transcriptional start andstop sequences, translational start and stop sequences, and enhancer oractivator sequences.

A “regulatable promoter” refers to a promoter that effects itsregulatory control over a nucleic acid sequence under specificenvironmental conditions. For example, an inducible promoter is one thatcauses expression of the operably linked polynucleotide under certainenvironmental conditions, for example, blue light inducible promoters(bli-4), and copper metallothionein gene (cmt). In a more specificexample, the glucoamylase A promoter (glaAp) induces expression in thepresence of maltose.

“Chimeric gene” or “heterologous nucleic acid construct”, as definedherein refers to a non-native gene (i.e., one that has been introducedinto a host) that may be composed of parts of different genes, includingregulatory elements. A chimeric gene construct for transformation of ahost cell is typically composed of a transcriptional regulatory region(promoter) operably linked to a heterologous protein coding sequence,or, in a selectable marker chimeric gene, to a selectable marker geneencoding a protein conferring antibiotic resistance to transformedcells. A typical chimeric gene of the present invention, fortransformation into a host cell, includes a transcriptional regulatoryregion that is constitutive or inducible, a protein coding sequence, anda terminator sequence. A chimeric gene construct may also include asecond DNA sequence encoding a signal peptide if secretion of the targetprotein is desired.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNAencoding a secretory leader is operably linked to DNA for a polypeptideif it is expressed as a preprotein that participates in the secretion ofthe polypeptide; a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence; or a ribosomebinding site is operably linked to a coding sequence if it is positionedso as to facilitate translation. Generally, “operably linked” means thatthe DNA sequences being linked are contiguous, and, in the case of asecretory leader, contiguous and in reading phase. However, enhancers donot have to be contiguous. Linking is accomplished by ligation atconvenient restriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice.

“Antisense” refers to sequences of nucleic acids that are complementaryto the coding mRNA nucleic acid sequence of a gene. A nucleotidesequence linked to a promoter in an “antisense orientation” is linked tothe promoter such that an RNA molecule complementary to the coding mRNAof the target gene is produced.

As used herein, the term “gene” means the segment of DNA involved inproducing a polypeptide chain, that may or may not include regionspreceding and following the coding region, e.g. 5′ untranslated (5′ UTR)or “leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons).

In general, nucleic acid molecules that encode cotA or an analog orhomologue thereof will hybridize, under moderate to high stringencyconditions to any one of the sequences provided herein as SEQ ID NO:1,3, 5, 13 or 14. However, in some cases a cotA-encoding nucleotidesequence is employed that possesses a substantially different codonusage, while the protein encoded by the cotA-encoding nucleotidesequence has the same or substantially the same amino acid sequence asthe native protein. For example, the coding sequence may be modified tofacilitate faster expression of cotA in a particular prokaryotic oreukaryotic expression system, in accordance with the frequency withwhich a particular codon is utilized by the host.

A nucleic acid sequence is considered to be “selectively hybridizable”to a reference nucleic acid sequence if the two sequences specificallyhybridize to one another under moderate to high stringency hybridizationand wash conditions. Hybridization conditions are based on the meltingtemperature (T_(m)) of the nucleic acid binding complex or probe. Forexample, “maximum stringency” typically occurs at about T_(m)-5° C. (5°below the T_(m) of the probe); “high stringency” at about 5-10° belowthe T_(m); “intermediate stringency” at about 10-20° below the T_(m) ofthe probe; and “low stringency” at about 20-25° below the T_(m).Functionally, maximum stringency conditions may be used to identifysequences having strict identity or near-strict identity with thehybridization probe; while high stringency conditions are used toidentify sequences having about 80% or more sequence identity with theprobe.

Moderate and high stringency hybridization conditions are well known inthe art (see, for example, Sambrook, et al, 1989, Chapters 9 and 11, andin Ausubel, F. M., et al, 1993, expressly incorporated by referenceherein). An example of high stringency conditions includes hybridizationat about 42° C. in 50% formamide, 5×SSC, 5×Denhardt's solution, 0.5% SDSand 100 μg/ml denatured carrier DNA followed by washing two times in2×SSC and 0.5% SDS at room temperature and two additional times in0.1×SSC and 0.5% SDS at 42° C.

The term “% homology” is used interchangeably herein with the term “%identity” herein and refers to the level of nucleic acid or amino acidsequence identity between the nucleic acid sequence that encodes cotA orthe cotA amino acid sequence, when aligned using a sequence alignmentprogram.

For example, as used herein, 80% homology means the same thing as 80%sequence identity determined by a defined algorithm, and accordingly ahomologue of a given sequence has greater than 80% sequence identityover a length of the given sequence. Exemplary levels of sequenceidentity include, but are not limited to, 80, 85, 90, 95, 98% or moresequence identity to a given sequence, e.g., the coding sequence forcotA, as described herein.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet. See, also, Altschul, S. F. et al.,1990 and Altschul, S. F. et al., 1997.

Sequence searches are typically carried out using the BLASTN programwhen evaluating a given nucleic acid sequence relative to nucleic acidsequences in the GenBank DNA Sequences and other public databases. TheBLASTX program is preferred for searching nucleic acid sequences thathave been translated in all reading frames against amino acid sequencesin the GenBank Protein Sequences and other public databases. Both BLASTNand BLASTX are run using default parameters of an open gap penalty of11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62matrix. [See, Altschul, et al., 1997.]

A preferred alignment of selected sequences in order to determine “%identity” between two or more sequences, is performed using for example,the CLUSTAL-W program in MacVector version 6.5, operated with defaultparameters, including an open gap penalty of 10.0, an extended gappenalty of 0.1, and a BLOSUM 30 similarity matrix.

In one exemplary approach, sequence extension of a nucleic acid encodingcotA may be may be carried out using conventional primer extensionprocedures as described in Sambrook et al., supra, to detect cotAprecursors and processing intermediates of mRNA that may not have beenreverse-transcribed into cDNA and/or to identify ORFs that encode a fulllength protein.

A nucleotide sequence encoding a cotA-type polypeptide, e.g., cot1 fromNeurospora crassa, can also be used to construct hybridization probesfor mapping the gene which encodes a cotA polypeptide and for furthergenetic analysis. Screening of a cDNA or genomic library with theselected probe may be conducted using standard procedures, such asdescribed in Sambrook et al., 1989). Hybridization conditions, includingmoderate stringency and high stringency, are provided in Sambrook etal., supra.

The probes or portions thereof may also be employed in PCR techniques togenerate a pool of sequences for identification of closely related cotAsequences. When cotA sequences are intended for use as probes, aparticular portion of a cotA encoding sequence, for example a highlyconserved portion of the coding sequence may be used.

For example, a cotA nucleotide sequence may be used as a hybridizationprobe for a cDNA library to isolate genes, for example, those encodingnaturally-occurring variants of cotA from other filamentous fungalspecies, which have a desired level of sequence identity to any one ofthe cotA nucleotide sequences disclosed in FIG. 1, 3, 5, 11 or 12 (SEQID NO:1, 3, 5, 13 or 14, respectively). Exemplary probes have a lengthof about 20 to about 50 bases but can go as long as 250 bp.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention.

As used herein, the terms “transformed”, “stably transformed” or“transgenic” with reference to a cell means the cell has a non-native(heterologous) nucleic acid sequence integrated into its genome that ismaintained through two or more generations.

As used herein, the term “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell where the nucleicacid sequence may be incorporated into the genome of the cell (forexample, chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (for example,transfected mRNA).

As used herein, the term “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation. It follows thatthe term “cotA expression” refers to transcription and translation ofthe cotA gene, the products of which include precursor RNA, mRNA,polypeptide, post-translation processed polypeptide, and derivativesthereof, including cotA homologs from other fungal species. By way ofexample, assays for cotA expression include examination of fungalcolonies when exposed to the appropriate conditions, western blot forcotA protein, as well as northern blot analysis and reversetranscriptase polymerase chain reaction (RT-PCR) assays for cotA mRNA.

“Alternative splicing” is a process whereby multiple polypeptideisoforms are generated from a single gene, and involves the splicingtogether of nonconsecutive exons during the processing of some, but notall, transcripts of the gene. Thus a particular exon may be connected toany one of several alternative exons to form messenger RNAs. Thealternatively-spliced mRNAs produce polypeptides (“splice variants”) inwhich some parts are common while other parts are different.

By “host cell” is meant a cell that contains a vector and supports thereplication, and/or transcription or transcription and translation(expression) of the expression construct. Host cells for use in thepresent invention can be prokaryotic cells, such as E. coli, oreukaryotic cells such as filamentous fungal, yeast, plant, insect,amphibian, or mammalian cells. In general, host cells are filamentousfungal cells. Specifically, the present invention find A. nidulans, A.niger and T. reesei cells advantageous.

The terms “isolated” or “purified” as used herein refer to a nucleicacid or polypeptide that is removed from at least one component withwhich it is naturally associated.

As used herein, the terms “active” and “biologically active” refer to abiological activity associated with a particular protein, such as theenzymatic activity associated with a kinase. It follows that thebiological activity of a given protein refers to any biological activitytypically attributed to that protein by those of skill in the art.

The phrase “slowed growth morphology” means the cells exhibit a moreslowly growing phenotype than wild type cells. This is evidenced by amore compact colony appearance on solid growth medium. This morphologymay be accompanied by hyphal hyper-branching.

II. TARGET ORGANISMS

In this invention, the source of the polynucleotides that encode cotA isa filamentous fungus. As well as being the source, in a preferredembodiment, the host cell is also a filamentous fungus cell. Filamentousfungi include all filamentous forms of the subdivision Eumycota andOomycota. The filamentous fungi are characterized by vegetative myceliumcomposed of chitin, cellulose, glucan, chitosan, mannan, and othercomplex polysaccharides, with vegetative growth by hyphal elongation andcarbon catabolism that is obligately aerobic.

In the present invention, the filamentous fungal parent cell may be acell of a species of, but not limited to, Aspergillus, Humicola andTrichoderma. In one embodiment, the filamentous fungal parent cell is anAspergillus niger, or an Aspergillus nidulans cell. In a first aspect,the parent cell is an Aspergillus niger cell. In a second aspect, theparent cell is an Aspergillus nidulans cell. In third aspect, thefilamentous fungal parent cell is a Trichoderma reesei cell. In a fourthaspect, the filamentous fungal parent cell is Humicola grisea.

III. METHODS OF IDENTIFYING NOVEL SEQUENCES

It has been discovered that cotA-encoding polynucleotides sharesignificant identity at the 3′ terminus. This region encodes thecatalytic region of cotA. Thus, it is expected that cotA homologs fromother fungal species may be found through searching fungal genomes forhomologous sequences or by degenerate PCR cloning of the conservedregion. Open reading frames (ORFs) within a fungal genome are analyzedfollowing full or partial sequencing of the target organism (in thiscase, fungal) genome and are further analyzed using sequence analysissoftware, and by determining homology to known sequences in databases(public/private). Sequence searching and comparison techniques are wellknown and readily available via the World Wide Web.

In a one aspect of this invention, cotA homologs are discovered throughdegenerate PCR cloning. Useful primers include, but are not limited to,5′-GA T/C AT T/C AA A/G CCNGA T/C AA-3′ (SEQ ID NO:7) and 5′-TCNGGNGCG/T/A AT A/G TA A/G TC-3′ (SEQ ID NO:8). Other primers will be apparentto those of skill in the art upon review of the sequences listed in FIG.7. PCR conditions to optimize hybridization of degenerate primers togenomic DNA and subsequent amplification are well within the purview ofthose of skill in the art. Such conditions may be found in Ausubeland/or Sambrook.

Although genomic sequences can be discovered directly through PCRcloning, in a preferred method, a probe consisting of a partialpolynucleotide sequence is generated via PCR cloning. Typically thisprobe is less than 1000 base pairs, more preferably less than 750 basepairs, even more preferably less than 500 bp and most preferably lessthan 250 base pairs. In a particularly preferred embodiment, the probeis from about 241 to 269 base pairs in length (FIG. 13 (SEQ ID NO:15)and corresponds approximately to residues 1144-1405 of the N. crassacot-1 sequence.

IV. COTA POLYPEPTIDES AND NUCLEIC ACID MOLECULES ENCODING COTA

A. cotA Nucleic Acids

The nucleic acid molecules of the present invention include a codingsequence for A. niger cotA presented herein as SEQ. ID. NO: 13 or A.nidulans presented herein as SEQ. ID. NO: 14, naturally occurringallelic and splice variants, nucleic acid fragments, and biologicallyactive (functional) derivatives thereof, such as, amino acid sequencevariants of the native molecule and sequences which encode fusionproteins.

The nucleic acid molecules of the present invention include a partialnative coding sequence for cotA presented herein as SEQ. ID. NO:1, andhomologues thereof in other species (for example, SEQ ID NO:3 (cotA fromA. nidulans) and SEQ ID NO:5 (cotA from T. reesei)), naturally occurringallelic and splice variants, nucleic acid fragments, and biologicallyactive (functional) derivatives thereof, such as, amino acid sequencevariants of the native molecule and sequences which encode fusionproteins. The sequences, both full length and partial sequences, arecollectively referred to herein as “cotA-encoding nucleic acidsequences”.

A cotA nucleic acid sequence of this invention may be a DNA or RNAsequence, derived from genomic DNA, cDNA, mRNA, or may be synthesized inwhole or in part. The DNA may be double-stranded or single-stranded andif single-stranded may be the coding strand or the non-coding(antisense, complementary) strand. The nucleic acid sequence may becloned, for example, by isolating genomic DNA from an appropriatesource, and amplifying and cloning the sequence of interest using apolymerase chain reaction (PCR). Alternatively, nucleic acid sequencesmay be synthesized, either completely or in part, especially where it isdesirable to provide host-preferred sequences for optimal expression.Thus, all or a portion of the desired structural gene (that portion ofthe gene which encodes a polypeptide or protein) may be synthesizedusing codons preferred by a selected host, e.g., Aspergillus niger,Aspergillus nidulans or Trichoderma reesei.

Due to the inherent degeneracy of the genetic code, nucleic acidsequences other than the native form that encode substantially the sameor a functionally equivalent amino acid sequence may be used to cloneand/or express cotA-encoding nucleic acid sequences. Thus, for a givencotA-encoding nucleic acid sequence, it is appreciated that, as a resultof the degeneracy of the genetic code, a number of coding sequences canbe produced that encode a protein having the same amino acid sequence.For example, the triplet CGT encodes the amino acid arginine. Arginineis alternatively encoded by CGA, CGC, CGG, AGA, and AGG. Therefore it isappreciated that such substitutions in the coding region fall within thenucleic acid sequence variants covered by the present invention. Any andall of these sequence variants can be utilized in the same way asdescribed herein for the native form of a cotA-encoding nucleic acidsequence.

A “variant” cotA-encoding nucleic acid sequence may encode a “variant”cotA amino acid sequence which is altered by one or more amino acidsfrom the native polypeptide sequence, both of which are included withinthe scope of the invention. Similarly, the term “modified form of”,relative to cotA, means a derivative or variant form of the native cotAprotein-encoding nucleic acid sequence or the native cotA amino acidsequence.

Similarly, the polynucleotides for use in practicing the inventioninclude sequences which encode native cotA proteins and splice variantsthereof, sequences complementary to the native protein coding sequence,and novel fragments of cotA encoding polynucleotides.

In one general embodiment, a cotA-encoding nucleotide sequence has atleast 70%, preferably 80%, 85%, 90%, 95%, 98%, or more sequence identityto any one of the cotA coding sequences presented herein as SEQ IDNOs:1, 3 or 5.

In another embodiment, a cotA-encoding nucleotide sequence willhybridize under moderate to high stringency conditions to a nucleotidesequence that encodes a cotA protein. In a related embodiment, acotA-encoding nucleotide sequence will hybridize under moderate to highstringency conditions to any one of the nucleotide sequences presentedas SEQ ID NOs:1, 3 or 5.

It is appreciated that some nucleic acid sequence variants that encodecotA may or may not selectively hybridize to the parent sequence. By wayof example, in situations where the coding sequence has been optimizedbased on the degeneracy of the genetic code, a variant coding sequencemay be produced that encodes a cotA protein, but does not hybridize to anative cotA-encoding nucleic acid sequence under moderate to highstringency conditions. This would occur, for example, when the sequencevariant includes a different codon for each of the amino acids encodedby the parent nucleotide.

As will be further understood by those of skill in the art, in somecases it may be advantageous to produce nucleotide sequences possessingnon-naturally occurring codons. Codons preferred by a particulareukaryotic host (Murray, E. et al., 1989) can be selected, for example,to increase the rate of cotA protein expression or to producerecombinant RNA transcripts having desirable properties, such as alonger half-life, than transcripts produced from the naturally occurringsequence. Hence, a native cotA-encoding nucleotide sequence may beengineered in order to alter the coding sequence for a variety ofreasons, including but not limited to, alterations which modify thecloning, processing and/or expression of the cotA protein by a cell.

A cotA-encoding nucleotide sequence may be engineered in order to alterthe cotA coding sequence for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing and/orexpression of cotA by a cell.

Particularly preferred are nucleic acid substitutions, additions, anddeletions that are silent such that they do not alter the properties oractivities of the native polynucleotide or polypeptide.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., 1986;Zoller et al., 1987], cassette mutagenesis [Wells et al., 1985],restriction selection mutagenesis [Wells et al., 1986] or other knowntechniques can be performed on the cloned DNA to produce the cotApolypeptide-encoding variant DNA.

However, in some cases it may be advantageous to express variants ofcotA which lack the properties or activities of the native cotApolynucleotide or polypeptide. In such cases, mutant or modified formsof the native cotA-encoding nucleic acid sequence may be generated usingtechniques routinely employed by those of skill in the art. For example,in a preferred embodiment, a fragment of a cotA-encoding polynucleotideis transfected into a fungal host cell. The manufacture of fragments offull length genomic and/or coding sequences is well within the skill ofone in the art.

B. cotA Polypeptides

In one embodiment, the invention provides a truncated cotA polypeptide,having a polypeptide sequence comprising the sequence presented in FIG.2 (SEQ ID NO:2). In another embodiment, a cotA polypeptide of theinvention can be the mature cotA polypeptide, part of a fusion proteinor a fragment or variant of the cotA polypeptide.

In another embodiment, the invention provides a truncated cotApolypeptide, having a polypeptide sequence comprising the sequencepresented in FIG. 4 (SEQ ID NO:4). In another embodiment, a cotApolypeptide of the invention can be the mature cotA polypeptide, part ofa fusion protein or a fragment or variant of the cotA polypeptide.

In a third embodiment, the invention provides a truncated cotApolypeptide, having a polypeptide sequence comprising the sequencepresented in FIG. 6 (SEQ ID NO:6). In another embodiment, a cotApolypeptide of the invention can be the mature cotA polypeptide, part ofa fusion protein or a fragment or variant of the cotA polypeptide.

Ordinarily, a cotA polypeptide of the invention comprises a regionhaving at least 80, 85, 90, 95, 98% or more sequence identity to any oneof the cotA polypeptide sequences of FIG. 2, 4 or 6 (SEQ ID NO:2, 4 or6, respectively), using a sequence alignment program, as detailedherein.

Typically, a “modified form of” a native cotA protein or a “variant”cotA protein has a derivative sequence containing at least one aminoacid substitution, deletion or insertion, respectively.

Fragments and variants of any one of the cotA polypeptide sequences ofFIG. 2, 4 or 6 (SEQ ID NOs:2, 4 or 6, respectively), are also consideredto be a part of the invention. A fragment is a variant polypeptide whichhas an amino acid sequence that is entirely the same as part but not allof the amino acid sequence of the previously described polypeptides. Thefragments can be “free-standing” or comprised within a largerpolypeptide of which the fragment forms a part or a region, mostpreferably as a single continuous region. Preferred fragments arebiologically active fragments which are those fragments that mediateactivities of the polypeptides of the invention, including those withsimilar activity or improved activity or with a decreased activity. Alsoincluded are those fragments that antigenic or immunogenic in an animal,particularly a human. In this aspect, the invention includes (i)fragments of cotA, preferably at least about 20-100 amino acids inlength, more preferably about 100-200 amino acids in length, and (ii) apharmaceutical composition comprising cotA. In various embodiments, thefragment corresponds to the N-terminal domain of cotA or the C-terminaldomain of cotA.

cotA polypeptides of the invention also include polypeptides that varyfrom any one of the cotA polypeptide sequences of FIG. 2, 4 or 6 (SEQ IDNO:2, 4 or 6, respectively). These variants may be substitutional,insertional or deletional variants. The variants typically exhibit thesame qualitative biological activity as the naturally occurringanalogue, although variants can also be selected which have modifiedcharacteristics as further described below.

A “substitution” results from the replacement of one or more nucleotidesor amino acids by different nucleotides or amino acids, respectively.

An “insertion” or “addition” is that change in a nucleotide or aminoacid sequence which has resulted in the addition of one or morenucleotides or amino acid residues, respectively, as compared to thenaturally occurring sequence.

A “deletion” is defined as a change in either nucleotide or amino acidsequence in which one or more nucleotides or amino acid residues,respectively, are absent.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances.

Amino acid substitutions can be the result of replacing one amino acidwith another amino acid having similar structural and/or chemicalproperties, such as the replacement of a leucine with a serine, i.e.,conservative amino acid replacements. Insertions or deletions mayoptionally be in the range of 1 to 5 amino acids.

Substitutions are generally made in accordance with known “conservativesubstitutions”. A “conservative substitution” refers to the substitutionof an amino acid in one class by an amino acid in the same class, wherea class is defined by common physicochemical amino acid side chainproperties and high substitution frequencies in homologous proteinsfound in nature (as determined, e.g. by a standard Dayhoff frequencyexchange matrix or BLOSUM matrix). (See generally, Doolittle, R. F.,1986.)

A “non-conservative substitution” refers to the substitution of an aminoacid in one class with an amino acid from another class.

cotA polypeptide variants typically exhibit the same qualitativebiological activity as the naturally-occurring analogue, althoughvariants also are selected to modify the characteristics of the cotApolypeptide, as needed. For example, glycosylation sites, and moreparticularly one or more O-linked or N-linked glycosylation sites may bealtered or removed. Those skilled in the art will appreciate that aminoacid changes may alter post-translational processes of the cotApolypeptide, such as changing the number or position of glycosylationsites or altering the membrane anchoring characteristics.

Also included within the definition of cotA polypeptides are otherrelated cotA polypeptides. Thus, probe or degenerate polymerase chainreaction (PCR) primer sequences may be used to find other relatedpolypeptides. Useful probe or primer sequences may be designed to: allor part of the cotA polypeptide sequence, or sequences outside thecoding region. As is generally known in the art, preferred PCR primersare from about 15 to about 35 nucleotides in length, with from about 20to about 30 being preferred, and may contain inosine as needed. Theconditions for the PCR reaction are generally known in the art.

Covalent modifications of cotA polypeptides are also included within thescope of this invention. For example, the invention provides cotApolypeptides that are a mature protein and may comprise additional aminoor carboxyl-terminal amino acids, or amino acids within the maturepolypeptide (for example, when the mature form of the protein has morethan one polypeptide chain). Such sequences can, for example, play arole in the processing of a protein from a precursor to a mature form,allow protein transport, shorten or lengthen protein half-life, orfacilitate manipulation of the protein in assays or production. It iscontemplated that cellular enzymes are used to remove any additionalamino acids from the mature protein.

C. Anti-cotA Antibodies.

The present invention further provides anti-cotA antibodies. Theantibodies may be polyclonal, monoclonal, humanized, bispecific orheteroconjugate antibodies.

Methods of preparing polyclonal antibodies are known to the skilledartisan. The immunizing agent may be a cotA polypeptide or a fusionprotein thereof. It may be useful to conjugate the antigen to a proteinknown to be immunogenic in the mammal being immunized. The immunizationprotocol may be determined by one skilled in the art based on standardprotocols or routine experimentation. Alternatively, the anti-cotAantibodies may be monoclonal antibodies. Monoclonal antibodies may beproduced by cells immunized in an animal or using recombinant DNAmethods. [See, e.g., Kohler et al., 1975; U.S. Pat. No. 4,816,567].Antibodies to proteins have many uses well known to those of skill inthe art. Here, it is envisioned that antibodies to cotA are useful as acomponent of staining reagents to determine the expression of cotA infungal host cells among other uses that will be apparent to those ofskill.

V. EXPRESSION OF RECOMBINANT cotA AND cotA FRAGMENTS

This invention provides filamentous fungal host cells which have beentransduced, transformed or transfected with an expression vectorcomprising a cotA-encoding nucleic acid sequence. The cultureconditions, such as temperature, pH and the like, are those previouslyused for the parental host cell prior to transduction, transformation ortransfection and will be apparent to those skilled in the art.

In one approach, a filamentous fungal cell line is transfected with anexpression vector having a promoter or biologically active promoterfragment or one or more (e.g., a series) of enhancers which functions inthe host cell line, operably linked to a DNA segment encoding cotA, suchthat cotA is expressed in the cell line. In a preferred embodiment, theDNA sequences encode a partial cotA coding sequence. In anotherpreferred embodiment, the promoter is a regulatable one.

A. Nucleic Acid Constructs/Expression Vectors.

Natural or synthetic polynucleotide fragments encoding cotA(“cotA-encoding nucleic acid sequences”) may be incorporated intoheterologous nucleic acid constructs or vectors, capable of introductioninto, and replication in, a filamentous fungal cell. The vectors andmethods disclosed herein are suitable for use in host cells for theexpression of cotA. Any vector may be used as long as it is replicableand viable in the cells into which it is introduced. Large numbers ofsuitable vectors and promoters are known to those of skill in the art,and are commercially available. Appropriate cloning and expressionvectors for use in filamentous fungal cells are also described inSambrook et al., 1989, and Ausubel F M et al., 1989, expresslyincorporated by reference herein. The appropriate DNA sequence may beinserted into a plasmid or vector (collectively referred to herein as“vectors”) by a variety of procedures. In general, the DNA sequence isinserted into an appropriate restriction endonuclease site(s) bystandard procedures. Such procedures and related sub-cloning proceduresare deemed to be within the scope of knowledge of those skilled in theart.

Appropriate vectors are typically equipped with a selectablemarker-encoding nucleic acid sequence, insertion sites, and suitablecontrol elements, such as termination sequences. The vector may compriseregulatory sequences, including, for example, non-coding sequences, suchas introns and control elements, i.e., promoter and terminator elementsor 5′ and/or 3′ untranslated regions, effective for expression of thecoding sequence in host cells (and/or in a vector or host cellenvironment in which a modified soluble protein antigen coding sequenceis not normally expressed), operably linked to the coding sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, many of which are commercially available and/or aredescribed in Sambrook, et al., (supra).

Exemplary promoters include both constitutive promoters and induciblepromoters, examples of which include a CMV promoter, an SV40 earlypromoter, an RSV promoter, an EF-1α promoter, a promoter containing thetet responsive element (TRE) in the tet-on or tet-off system asdescribed (ClonTech and BASF), the beta actin promoter and themetallothienein promoter that can upregulated by addition of certainmetal salts. In one embodiment of this invention, glaA promoter is used.This promoter is induced in the presence of maltose. In a preferredembodiment, a promoter that is induced by maltose is used. Suchpromoters are well known to those of skill in the art.

The choice of the proper selectable marker will depend on the host cell,and appropriate markers for different hosts are well known in the art.Typical selectable marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, for example, ampicillin,methotrexate, tetracycline, neomycin (Southern and Berg, J., 1982),mycophenolic acid (Mulligan and Berg, 1980), puromycin, zeomycin, orhygromycin (Sugden et al., 1985). In a preferred embodiment, PyrG isused as a selectable marker.

A selected cotA coding sequence may be inserted into a suitable vectoraccording to well-known recombinant techniques and used to transform acell line capable of cotA expression. Due to the inherent degeneracy ofthe genetic code, other nucleic acid sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be used to clone and express cotA, as further detailed above.Therefore it is appreciated that such substitutions in the coding regionfall within the sequence variants covered by the present invention. Anyand all of these sequence variants can be utilized in the same way asdescribed herein for a parent cotA-encoding nucleic acid sequence.

Once the desired form of a cotA nucleic acid sequence, homologue,variant or fragment thereof, is obtained, it may be modified in avariety of ways. Where the sequence involves non-coding flankingregions, the flanking regions may be subjected to resection,mutagenesis, etc. Thus, transitions, transversions, deletions, andinsertions may be performed on the naturally occurring sequence.

The present invention also includes recombinant nucleic acid constructscomprising one or more of the cotA-encoding nucleic acid sequences asdescribed above. The constructs comprise a vector, such as a plasmid orviral vector, into which a sequence of the invention has been inserted,in a forward or reverse orientation.

Heterologous nucleic acid constructs may include the coding sequence forcotA, or a variant, fragment or splice variant thereof: (i) inisolation; (ii) in combination with additional coding sequences; such asfusion protein or signal peptide coding sequences, where the cotA codingsequence is the dominant coding sequence; (iii) in combination withnon-coding sequences, such as introns and control elements, such aspromoter and terminator elements or 5′ and/or 3′ untranslated regions,effective for expression of the coding sequence in a suitable host;and/or (iv) in a vector or host environment in which the cotA codingsequence is a heterologous gene.

A heterologous nucleic acid containing the appropriate nucleic acidcoding sequence, as described above, together with appropriate promoterand control sequences, may be employed to transform filamentous fungalcells to permit the cells to express cotA.

In one aspect of the present invention, a heterologous nucleic acidconstruct is employed to transfer a cotA-encoding nucleic acid sequenceinto a cell in vitro, with established cell lines preferred. Preferably,cell lines that are to be used as production hosts have the nucleic acidsequences of this invention stably integrated. Integration preferablyoccurs in the cotA locus but ectopic integration is useful as well. Itfollows that any method effective to generate stable transformants maybe used in practicing the invention.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,“Molecular Cloning: A Laboratory Manual”, Second Edition (Sambrook,Fritsch & Maniatis, 1989), “Animal Cell Culture” (R. I. Freshney, ed.,1987); “Current Protocols in Molecular Biology” (F. M. Ausubel et al.,eds., 1987); and “Current Protocols in Immunology” (J. E. Coligan etal., eds., 1991). All patents, patent applications, articles andpublications mentioned herein, both supra and infra, are herebyexpressly incorporated herein by reference.

B. Host Cells and Culture Conditions for Regulatable Expression.

Thus, the present invention provides cell lines comprising cells whichhave been modified, selected and cultured in a manner effective toresult in regulatable expression of cotA relative to the correspondingnon-transformed parental cell line.

Examples of parental cell lines which may be treated and/or modified forregulatable cotA expression include, but are not limited to filamentousfungal cells. Examples of appropriate primary cell types for use inpracticing the invention include, but are not limited to, Aspergillusand Trichoderina.

cotA expressing cells are cultured under conditions typically employedto culture the parental cell line. Generally, cells are cultured in astandard medium containing physiological salts and nutrients, such asstandard RPMI, MEM, IMEM or DMEM, typically supplemented with 5-10%serum, such as fetal bovine serum. Culture conditions are also standard,e.g., cultures are incubated at 37° C. in stationary or roller culturesuntil desired levels of cotA expression are achieved.

Preferred culture conditions for a given cell line may be found in thescientific literature and/or from the source of the cell line such asthe American Type Culture Collection. Typically, after cell growth hasbeen established, the cells are exposed to conditions effective to causeor inhibit the expression of cotA and truncated cotA.

In the preferred embodiments, where a cotA coding sequence is under thecontrol of an inducible promoter, the inducing agent, e.g. acarbohydrate, metal salt or antibiotics, is added to the medium at aconcentration effective to induce cotA expression.

C. Introduction of a cotA-Encoding Nucleic Acid Sequence into HostCells.

The invention further provides cells and cell compositions which havebeen genetically modified to comprise an exogenously providedcotA-encoding nucleic acid sequence. A parental cell or cell line may begenetically modified (i.e., transduced, transformed or transfected) witha cloning vector or an expression vector. The vector may be, forexample, in the form of a plasmid, a viral particle, a phage, etc, asfurther described above. In a preferred embodiment, a plasmid is used totransfect a filamentous fungal cell.

Various methods may be employed for delivering an expression vector intocells in vitro. Methods of introducing nucleic acids into cells forexpression of heterologous nucleic acid sequences are also known to theordinarily skilled artisan, including, but not limited toelectroporation; nuclear microinjection or direct microinjection intosingle cells; bacterial protoplast fusion with intact cells; use ofpolycations, e.g., polybrene or polyornithine; membrane fusion withliposomes, lipofectamine or lipofection-mediated transfection; highvelocity bombardment with DNA-coated microprojectiles; incubation withcalcium phosphate-DNA precipitate; DEAE-Dextran mediated transfection;infection with modified viral nucleic acids; and the like. In addition,heterologous nucleic acid constructs comprising a cotA-encoding nucleicacid sequence can be transcribed in vitro, and the resulting RNAintroduced into the host cell by well-known methods, e.g., by injection.

In a preferred embodiment, the expression vector comprising a truncatedcotA and an appropriate promoter is constructed such that the promoterand cotA sequence integrates in the cotA locus. This is accomplished viaa single recombination event within the cotA locus. In a more preferredembodiment, the expression vector is constructed such that a doublerecombination event occurs. The vector comprises a stretch of nucleicacid that is complementary to a stretch of nucleic acid in the cotAlocus upstream from the cotA coding sequence. The other site ofcomplementary DNA occurs in the coding region. Upon integration, twocrossover events occur so that only the appropriate promoter and thetruncated cotA sequence are inserted into the cotA locus instead of theentire expression vector.

Following introduction of a heterologous nucleic acid constructcomprising the coding sequence for cotA, the genetically modified cellscan be cultured in conventional nutrient media modified as appropriatefor activating promoters, selecting transformants or amplifyingexpression of a cotA-encoding nucleic acid sequence. The cultureconditions, such as temperature, pH and the like, are those previouslyused for the host cell selected for expression, and will be apparent tothose skilled in the art.

The progeny of cells into which such heterologous nucleic acidconstructs have been introduced are generally considered to comprise thecotA-encoding nucleic acid sequence found in the heterologous nucleicacid construct.

VI. ANALYSIS OF COTA NUCLEIC ACIDS AND PROTEINS

In order to evaluate the expression of cotA by a cell line that has beentransformed with a cotA-encoding nucleic acid construct, assays can becarried out at the protein level, the RNA level or by use of functionalbioassays particular to growth characteristics of the transfected cellline.

By way of example, the production and/or expression of cotA may bemeasured in a sample directly, for example, by microscopic examinationof transfected cells. Filamentous fungal cells that have beentransfected with cotA under the control of an inducible promoter exhibitslowed and more compact growth compared to parental fungal cells whenexposed to the compound that induces expression. Nucleic acid-basedassays for determining the expression of cotA include, but are notlimited to, northern blotting to quantitate the transcription of mRNA,dot blotting (DNA or RNA analysis), RT-PCR (reverse transcriptasepolymerase chain reaction), or in situ hybridization, using anappropriately labeled probe (based on the nucleic acid coding sequence)and conventional Southern blotting.

Alternatively, protein expression, may be evaluated by immunologicalmethods, such as immunohistochemical staining of cells, tissue sectionsor immunoassay of tissue culture medium, e.g., by western blot or ELISA.Such immunoassays can be used to qualitatively and quantitativelyevaluate expression of cotA. The details of such methods are known tothose of skill in the art and many reagents for practicing such methodsare commercially available.

A purified form of cotA is typically used to produce either monoclonalor polyclonal antibodies specific to the expressed protein for use invarious immunoassays. (See, e.g., Harlow and Lane, 1988). Exemplaryassays include ELISA, competitive immunoassays, radioimmunoassays,western blot, indirect immunofluorescent assays and the like. Ingeneral, commercially available antibodies and/or kits may be used forthe quantitative immunoassay of the expression level of known types ofproteins.

VII. ISOLATION AND PURIFICATION OF RECOMBINANT COTA PROTEIN

In general, a cotA protein produced in a filamentous fungal cell is notsecreted into the medium and therefore must be purified from celllysates. This can be accomplished by techniques routine employed bythose of skill in the art.

Typically, after removal of cell debris, the lysate comprising cotAprotein is fractionated to segregate proteins having selectedproperties, such as binding affinity to particular binding agents, e.g.,antibodies or receptors; or which have a selected molecular weightrange, or range of isoelectric points.

Once expression of a given cotA protein is achieved, the cotA proteinthereby produced is purified from the cells or cell culture. Exemplaryprocedures suitable for such purification include the following:antibody-affinity column chromatography, ion exchange chromatography;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; and gel filtration using, e.g., SephadexG-75. Various methods of protein purification may be employed and suchmethods are known in the art and described e.g. in Deutscher, 1990;Scopes, 1982. The purification step(s) selected will depend, e.g., onthe nature of the production process used and the particular proteinproduced.

VIII. UTILITY OF THE COTA POLYPEPTIDES AND NUCLEIC ACIDS OF THISINVENTION

From the foregoing, it can be appreciated that cells transformed withcotA under the control of an inducible promoter grow more slowly inconditions in which cotA is expressed. By retarding the growth of fungalcell cultures, fermenter cultures of such cells can be maintained forlonger periods of time. Because fermenter cultures are maintained forlonger periods, expressed protein levels can be maintained for longerperiods of time. Thus, elevated concentrations of expressed protein canbe achieved. As would be obvious to one of skill, this would lead tolower production costs.

For production of a desired protein in a fungal host cell, an expressionvector comprising at least one copy of nucleic acid encoding a desiredprotein is transformed into the recombinant host cell comprising nucleicacid encoding a protein associated with hyphal growth and cultured underconditions suitable for expression of the protein. Examples of desiredproteins include enzymes such as hydrolases including proteases,cellulases, amylases, carbohydrases, and lipases; isomerases such asracemases, epimerases, tautomerases, or mutases; transferases, kinasesand phophatases along with proteins of therapeutic value.

Thus, the present invention is particularly useful in enhancing theintracellular and/or extracellular production of proteins. The proteinmay be homologous or heterologous. Proteins that may produced by theinstant invention include, but are not limited to, hormones, enzymes,growth factors, cytokines, antibodies and the like.

Enzymes include, but are not limited to, hydrolases, such as protease,esterase, lipase, phenol oxidase, permease, amylase, pullulanase,cellulase, glucose isomerase, laccase and protein disulfide isomerase.

Hormones include, but are not limited to, follicle-stimulating hormone,luteinizing hormone, corticotropin-releasing factor, somatostatin,gonadotropin hormone, vasopressin, oxytocin, erythropoietin, insulin andthe like.

Growth factors are proteins that bind to receptors on the cell surface,with the primary result of activating cellular proliferation and/ordifferentiation. Growth factors include, but are not limited to,platelet-derived growth factor, epidermal growth factor, nerve growthfactor, fibroblast growth factors, insulin-like growth factors,transforming growth factors and the like.

Cytokines are a unique family of growth factors. Secreted primarily fromleukocytes, cytokines stimulate both the humoral and cellular immuneresponses, as well as the activation of phagocytic cells. Cytokinesinclude, but are not limited to, colony stimulating factors, theinterleukins (IL-1 (α and β), IL-2 through IL-13) and the interferons(α, β and γ).

Human Interleukin-3 (IL-3) is a 15 kDa protein containing 133 amino acidresidues. IL-3 is a species specific colony stimulating factor whichstimulates colony formation of megakaryocytes, neutrophils, andmacrophages from bone marrow cultures.

Antibodies include, but are not limited to, immunoglobulins from anyspecies from which it is desirable to produce large quantities. It isespecially preferred that the antibodies are human antibodies.Immunoglobulins may be from any class, i.e., G, A, M, E or D.

EXAMPLES

The following examples are submitted for illustrative purposes only andshould not be interpreted as limiting the invention in any way.

Example 1 Isolation of a Truncated cotA Polynucleotide from Aspergillusniger

Based on an alignment of cot1 from N. crassa, TB3 (a Colletotrichumhomologue), KNQ_(—)1/Cbk1p, a related kinase in S. cerevisiae, and Homosapiens DMK, degenerate oligonucleotides were designed against 2conserved regions of the coding sequence.

(SEQ ID NO: 7) DIKPDN (5′ forward primer) 5′-GA T/C AT T/C AA A/G CCNGAT/C AA-3′ (SEQ ID NO: 8) EPAIYD (3′ reverse primer) 5′-TCNGGNGC G/T/A ATA/G TA A/G TC-3′

Using routine PCR conditions and genomic A. niger DNA, a 241 internalfragment was produced. This fragment was sequenced and found to haveclosest homology to cot-1 of N. crassa. This fragment was used to probedigested A. niger genomic DNA on a Southern blot according to routinemethods. A 6.5 kb band from a HindIII digest hybridized with the probe.

A. niger genomic DNA was digested with HindIII, recircularized andligated. This circularized DNA was subjected to inverse PCR usingoligonucleotides designed from the nucleotide sequence of the 241 bpregion homologous to cot-1.

INV3′ (reverse primer) 5′ACGTCGAGITCTTCAGC 3′ (SEQ ID NO: 9)INV5′ (forward primer) 5′GCGATCAACCTGACAGT 3′ (SEQ ID NO: 10)

A 6.5 kb fragment produced from the inverse PCR reaction was insertedinto the cloning vector pCR® 2.1. The resulting construct, pPOL, wassequenced. The sequence data allowed orientation of the A. niger cotAwithin the 6.5 kb fragment. The selected open reading frame of A. nigerwas aligned with related kinases (See FIG. 7).

As can be seen from FIG. 7, the 6.5 kb fragment contains an open readingframe of approximately 500 amino acids or of 10.5 kb. Alignment of theORF of the A. niger homologue with cot-1 indicated that about 50 aminoacids or 150 base pairs from the C or 3′ terminal were missing from thecoding region.

Example 2 Expression of Truncated A. niger cotA

1.4 kb of the 5′ coding region of cotA under the control of the glaApromoter was inserted into the expression vector pGRT-pyrG1 (Ward, etal, Appl Microbiol and Biotech. 39:738-743 (1993)) to examine the effectof regulated expression of cotA on the growth morphology of A. niger.glaAp is induced by maltose and repressed by xylose. The resultingplasmid, pSMB5, was used to transform an A. niger pyrG-recipient. SeeFIG. 8 for schematic of transformation. Pyr+ transformants were selectedon minimal medium with maltose as the sole carbon source and screenedfor growth morphology on xylose. Transformants that showed restrictedgrowth on xylose but that grew well on maltose, were analyzed bySouthern hybridization. In one colony of transformants (SMB540),integration of the plasmid occurred at the cotA locus, in others,ectopic integration took place.

Parental A. niger cotA strains were compared to strains carrying theglaAp-cotA fusion after growth on different C-sources, to regulateexpression of glaAp. Morphological changes occurred only duringrepression of cotA expression, with YEPX more repressing than MM+1%xylose. When cotA+ and glaAp-cotA strains were grown on maltose(non-repressing) then no morphological difference was seen between thestrains.

Example 3 Truncated cotA in the Antisense Orientation

To determine what effect disruption of cotA would have on the growth ofA. niger, an A. niger strain was transformed with cotA under the controlof glaAp as above, except the cotA sequence was in the antisenseorientation.

As can be seen in FIG. 9, the morphology of the transformants is veryslow growing and compact with very long branches.

Example 4 Point Mutation in the cotA Locus

From the literature, it is known that in N. crassa, a single mutation inthe cot-1 locus creates the temperature sensitive hyperbranchingphenotype. In cot-1, a histidine naturally occurs at position 352 (seeFIG. 7). The cot-1 mutation is caused by a switch to arginine at thisposition.

Site directed mutagenesis can be used to manufacture the same mutationin the cotA coding sequence of A. niger. Using techniques very similarto those described above, the cotA coding sequence with the pointmutation as well as an inducible promoter can be integrated into thecotA locus or ectopically. It is expected that, when induced, themutation will cause the slow growth morphology described above

Example 5 Isolation of Truncated Cot-1 from Trichoderma reesei

Using degenerate PCR, a 264 base pair cot-1 nucleic acid sequence wasisolated from genomic T. reesei genomic DNA. The forward primer was 5′GA T/C AT T/C AA A/G CC A/G/C/T GA A/C AA-3′ (SEQ ID NO:11) and thereverse primer was 5′ TC A/G/C/T GG A/C/G/T GC G/T AT A/G TA A/G TC-3′(SEQ ID NO:12).

The internal cot-1 fragment is shown in FIG. 5 (SEQ ID NO:5) and thetranslated sequence is shown in FIG. 6 (SEQ ID NO:6).

1. An isolated polynucleotide that encodes or is fully complementary toa sequence that encodes a cotA polypeptide having serine/threoninekinase activity selected from the group consisting of: a nucleic acidsequence that encodes or is fully complementary to a sequence thatencodes a cotA polypeptide having at least 95% sequence identity to theamino acid sequence presented as SEQ ID NO:2 (FIG. 2), a nucleic acidsequence that encodes or is fully complementary to a sequence thatencodes a cotA polypeptide having the amino acid sequence presented asSEQ ID NO:2 (FIG. 2), and the nucleic acid sequence presented as SEQ IDNO:1 (FIG. 1) or the full complement thereof.
 2. An expression cassettecomprising a promoter operatively linked to a polynucleotide of claim 1.3. The expression cassette of claim 2, wherein the promoter isinducible.
 4. The expression cassette of claim 3, wherein the promoteris selected from the group consisting of glucoamylase gene promoter,blue light inducible promoters (bli-4), and copper metallothionein gene(cmt) promoter.
 5. The expression cassette of claim 4, wherein thepromoter is the glucoamylase A (glaA) promoter.
 6. An expression vectorcomprising the expression cassette of claim 2.